Purification and properties of phenolic acid decarboxylase from Candida guilliermondii

Abstract

A heat-labile phenolic acid decarboxylase from Candida guilliermondii (an anamorph of Pichia guilliermondii) was purified to homogeneity by simple successive column chromatography within 3 days. The molecular mass was 20 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa by gel-filtration chromatography, suggesting that the purified enzyme is a homodimer. The optimal pH and temperature were approximately 6.0 and 25°C. Characteristically, more than 50% of the optimal activity was observed at 0°C, suggesting that this enzyme is cold-adapted. The enzyme converted p-coumaric acid, ferulic acid, and caffeic acid to corresponding products with high specific activities of approximately 600, 530, and 46 U/mg, respectively. The activity was stimulated by Mg(2+) ions, whereas it was completely inhibited by Fe(2+), Ni(2+), Cu(2+), Hg(2+), 4-chloromericuribenzoate, N-bromosuccinimide, and diethyl pyrocarbonate. The enzyme was inducible and expressed inside the cells moderately by ferulic acid and p-coumaric acid and significantly by non-metabolizable 6-hydroxy-2-naphthoic acid.

Theoretical study of the reaction mechanism of phenolic acid decarboxylase

“…No experimental rate constant ( k cat ) has been measured for Bs PAD. However, k cat values for PCA decarboxylation catalyzed by several PAD‐type enzymes from other organisms have been reported to be between 113 s −1 and 6000 s −1 , corresponding to energy barriers of about 12–15 kcal·mol −1 . Thus, the calculated overall barrier of 16 kcal·mol −1 obtained here (Fig.…”

A Two-Step Bioconversion Process for Canolol Production from Rapeseed Meal Combining an Aspergillus niger Feruloyl Esterase and the Fungus Neolentinus lepideus

“…Non-oxidative microbial decarboxylation of phenolic compounds to vinyl derivatives has been described historically for ferulic acid (4-hydroxy-3-methoxycinnamic acid) in the residues of fermented products including beer and whisky [ 24 ]. This microbial activity involves an intracellular enzyme called phenolic acid decarboxylase (PAD), described in bacilli bacteria and some yeasts till now [ 25 , 26 , 27 ]. The substrate specificity of the native microbial PADs was described only for ferulic, p -coumaric and caffeic acids, in the decreasing order.…”

In situ oxidation of canola meal sinapic acid by horseradish peroxidase (type II) and tyrosinase

“…Decarboxylation of phenolic compounds to corresponding vinyl derivatives using enzymes has been applied for the fermentation industries such as beer and whisky residues for decades (Steinke & Paulson, ). However, the phenolic acid decarboxylase only catalyzes the reactions of specific substrates namely ferulic acid, caffeic acid and p‐coumaric acid (Godoy, Martínez, Carrasco, & Ganga, ; Huang et al, ). In our experiment, no canolol was found after the oxidation of sinapic acid, instead compounds 1 and 2 were the only oxidative products detected.…”

“…In our previous study, canola seed was heated using conventional oven or microwave energy to trigger the decarboxylation of sinapic acid (Mayengbam, Khattab, & Thiyam‐Holländer, ), and the main product from this process was shown to be canolol (Khattab et al, ; Zago et al, ). Recently, bioconversion process of phenolic acids has been developed and it is an environmental friendly and time‐saving alternative to heat treatment (Huang et al, ; Morley, Grosse, Leisch, & Lau, ). Unlike heat treatment, the products of enzyme reactions are very specific and highly reproducible with a lower probability of formation of unwanted side products.…”